The main objective of the proposed studies is to examine the biochemical mechanisms whereby the cytotoxicity of the DNAalkylating agent melphalan (L-phenylalanine mustard, LPAM) to human melanoma cells is enhanced by the coadministration of two agents, doxorubicin (DOX) and carmustine (BCNU) . The observation of a decrease in the IC(50) for LPAM cytotoxicity in DOX+BCNU-exposed cells to 1/55th of the cytotoxic IC(50) in control cells may be partially explained by the fact that these two agents act syner- gistically to deplete intracellular glutathione (GSH) . It appears, however, that additional biochemical mechanisms may be involved. In order to examine and elucidate mechanisms both related and possibly unrelated to GSH depletion, we propose the following studies: 1) examine LPAM cytotoxicity after exposure to DOX+BCNU in a number of cultured human melanoma cell lines; 2) investigate the role of GSH depletion in potentiating this cytotoxicity; 3) investigate the effects of DOX+BCNU exposure on the cellular disposition of LPAM; 4) investigate the effects of DOX+BCNU exposure on the formation of covalent adducts of LPAM with cellular DNA, and the subsequent development of interstrand DNA cross-links; 5) examine cellular calcium levels and the extent of protein thiol oxidation after DOX+BCNU exposure, focussing on the possible correlation of these early toxic events with GSH depletion and LPAM cytotoxicity; and 6) examine the effects of DOX+BCNU on LPAM cytotoxicity in vivo, using xenografts of these same melanoma cell lines in athymic mice. It is hoped that elucidation of these biochemical mechanisms of cytotoxicity enhancement will eventually aid the design of practical methods for enhancing the susceptibility of human melanoma, a neoplasm that is notoriously intractable to treatment by chemotherapy, to the cytotoxic effects of LPAM or other alkylating agents in vivo.